1. Field of Invention
This invention relates to treatment of human beings for conditions associated with deficiencies in the N-6-trimethyl-L-lysine (TML) pathway affecting biosynthesis of carnitine. Such conditions include: (1) seizures, myoclonic seizures, epilepsy, refractory epilepsy, hyperkinetic movements, or tremors of the hands or feet, (2) a state of ataxia, (3) accumulation of neuronal autofluorescent storage bodies in lysosomes or neurons, or regression of motor development, and (4) low alertness, dementia, or mental retardation.
2. Description of Related Art
All material referenced in the prior provisional and non-provisional applications are hereby incorporated by reference.
Batten disease is named after the British pediatrician who first described it in 1903. It is also known as Spielmeyer-Vogt-Sjogren-Batten disease. The disease progresses and strikes without warning. The first signs of Batten Disease may be visual impairment and seizures. As the disease progresses, visual impairment leads to blindness and myoclonic seizures become more frequent and intense. A child with Batten Disease will also have a marked decline in cognitive function, noticeable personality and behavioral changes, a loss of communication skills, a loss of motor skills, apparent plasticity, facial grimacing and abnormal body movements. Thus Batten Disease leads to a vegetative state and is ultimately fatal (NINDS Batten Disease Information Page at http://www.ninds.nih.gov/disorders/batten/batten.htm, found in the parent application in Appendix A).
The parent application described the symptoms and common as well as distinct characteristics of the spectrum of NCL group, including the following: Batten Disease, Santavuori disease, Late-Infantile Neuronal Ceroid Lipofuscinoses (LINCL), Speilmeyer-Sjogren disease, Kuf disease, Parry disease, Bernheimer-Seitelberger syndrome, Bielschowsky amaurotic idiocy, Bielschowsky disease, Jansky-Bielschowsky disease, Seitelberger disease, late infantile amaurotic idiocy, late infantile Batten disease, subacute late infantile neuronal ceroid-lipofuscinosis, Zeman-Dyken-Lake-Santavuori-Savukoski disease.
At the genetic level, the neuronal ceroid lipofuscinoses (NCL's) result from mutations in at least eight genes, and these mutations are responsible for causing the various expressions of the neurodegenerative diseases collectively identified as NCLs. A summary background of these mutations and a survey of the background reference literature were given in the parent application. See Table A by Gene Locus.
TABLE ANeuronal Ceroid Lipofuscinosis - Summary of SymptomsSYMPTOMSCLN1CLN2CLN3CLN4CLN5CLN6CLN7CLN8CLN9CLN10DementiaYesYesYesYesYesYesSeizuresYesYesYesYesYesYesYesYesYesyes(hyperkneticmovements,hand/feettremors)Progressive VisualYesYesYesYesYesYesYesNoYesnewborn infantFailureMental RetardationYesYesYesYesYesYesYesYesLoss Of SpeechYesYesYesYesYesYesyesRegression of MotorYesYesYesYesYesyesDevelopmentAtaxiaYesYesYesYesYesYesyesMuscularYesYesYesYesyesHypotonia/DystoniaMicrocephalyYesOptic Atrophy/MacularYesYesYesYesDegenerationRetinitis PigmentosaMyoclonusYesYesYesYesYesYesNoCerebellar AtrophyYesYesYesYesyesQuadraparesisYesRefractory EpilepsyYesBehavioral InvolvementYesYes(Anger Outburst, PhysicalViolence)CLN1 (Infantile)CLN2 (Late Infantile)CLN3 (Juvenile)CLN4a (Kufs Disease)CLN5 (Late Infantile, Finnish Variant)CLN6 (Late Infantile, Variant, Included, Variable age at onset)CLN7 Late Infantile; allele to Northern EpilepsyCLN8CLN8 (Northern Epilepsy Variant)CLN9CLN10 (Cathepsin D-Deficient, CongenitTable A Notes:1. According to Mole et al.. 2005, the clinical course of the NCL's include progressive dementia, seizures, and progressive visual failure (Full text available at http://www.springerlink.com/content/xu2406100j81034w/fulltext.pdf ).2. Obviously, a ‘yes’ means that the symptom is a characteristic of the disease. A ‘NO’ means that the OMIM synopsis from clearly stated that the specific symptom is NOT characteristic of that particular NCL. An empty space for a particular symptom does not necessarily preclude it from being part of the characteristics of that particular NCL; it was not mentioned specifically in the OMIM synopsis. For instance, CLN6 is an LINCL(CLN2) variant. It did not specificallymention Mental Retardation or Loss of Speech or Cerebellar Atrophy; but it would be hard to believe that Mental Retardation/Loss of Speech/Cerebellar would not be part of the continuum.3. References:CLN1 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=256730CLN2 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204500CLN3 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204200CLN4a http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204300CLN5 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=256731CLN6 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=601780CLN7 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=610951CLN8 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=600143CLN8 (northern epilepsy variant) http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=610003CLN9 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=609055CLN10 http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=610127
Neuronal ceroid-lipofuscinosis are a group of inherited progressive neurometabolic diseases, previously considered as several separate syndromic entities, with considerable variability in clinical, pathological manifestations, and genetic findings. All diseases in this group are characterized by abnormal storage of the autofluorescent proteolipopigments in neuronal and other formulas with an average incidence estimated at 1:5/100,000. Abnormal proteins occur in the lysosomes, due to defects in lysosomal proteases or related enzymes or in other words, defective lysosomal proteolysis. Abnormal accumulation of proteins in various tissuess is not only responsible for the Batten disease, but it is also well known that it is responsible for other well known disorders, such as Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington disease. In all these diseases, including Batten disease, undegraded proteins cause loss of neuronal cells.
NCLs are also classified on the basis of specific gene association and genetic features of the disease, age of onset, clinical manifestations, and pathological changes. The common characteristics in all of these disorders is accumulation of autofluorescent storage material in all tissues, but more pronounced in central nervous system. They are listed sequentially.
Neuronal ceroid-lipofuscinosis type 1 (CLN-1) Synonyms: Hagberg-Santavuori disease, Haltia-Santavuori disease, Santavuori disease, and several other synonyms. It is characterized by rapid deterioration with psychomotor retardation, loss of speech, seizures, ataxia, blindness, hypotonia, microcephaly, and occasional convulsions. Mapped to chromosome 1p32. The gene is known to be responsible for the making of palmitoyl-protein thioesterase. It is transmitted as an autosomal recessive trait. J. Vesa, E. Hellsten, L. A. Verkruyse, L. A. Camp, J. Rapola, P. Santavuori, S. L. Hofmann, L. Peltonen, Nature 376, 584-587,1995, E. Hellsten, J. Vesa, V. M. Olkkonen, A. Jalanko, L. Peltonen, EMBO J., 15, 5240-5245, 1996.
Neuronal ceroid-lipofuscinosis type 2 (CLN-2) Synonyms: Bernheimer-Seitelberger syndrome, Bielschowsky amaurotic idiocy, Bielschowsky disease, Jansky-Bielschowsky disease, Seitelberger disease, late infantile amaurotic idiocy, late infantile Batten disease, late infantile neuronal lipofuscinosis (LINCL), subacute late infantile neuronal ceroid-lipofuscinosis. This is the second most common variant with a subacute course after onset in infancy or early childhood characterized by refractive epilepsy, mental regression, ataxia, visual loss, and progressive deterioration. Mapped to chromosome 11p15. The CLN-2, the classical late infantile neuronal ceroid lipofuscinosis has been associated with mutation in a lysosomal protein. D. E. Sleat, R. J. Donnelly, H. Lackland, C.-G. liu, I. Sohar, R. K. Pullarkat and P. Lobel, Science 277, 1802-1805, 1997.
Neuronal ceroid-lipofuscinosis type 3 (CLN-3) Synonyms: Batten syndrome (BTS) Batten-Mayou syndrome, Batten-Spielmeyer-Vogt disease, Spielmeyer-Sjogren syndrome, Spielmeyer-Vogt-Batten disease, Spielmeyer-Vogt-Sjogren disease, Stock-Spielmeyer-Vogt syndrome, Vogt-Spielmeyer disease, chronic juvenile neuronal ceroid-lipofuscinosis (JNCL), juvenile amaurotic family idiocy, juvenile amaurotic idiocy, juvenile Batten disease, juvenile cerebrorenal degeneration, juvenile neuronal lipofuscinosis (JNCL), juvenile onset neuronal ceroid-lipofuscinosis, and pigmentary retinal neuronal heredodegeneration. The most commonly occurring variant has a chronic course after juvenile onset with an estimated incidence of 1:25,000. The first symptom is usually visual failure, which takes place between the ages of 4 and 15 years. The early symptoms are followed by epilepsy and progressive physical and mental deterioration. Batten disease gene maps to chromosome 16p12.1.56. Further it has been shown that the Batten disease protein CLN3P localizes into membrane lipid raits, which are detergent-resistant membranes (Rakheja D., Narayan S. B., Pastor J. V., Bennett M. J., Biochemical & Biophysical Research Communications. 317(4):988-991, 2004. Studies have been carried out on the intracellular trafficking of CLN3 protein, underlying the childhood neurodegenerative conditions of Batten disease (Mao Q., Xia H., Davidson B. L., FEBS Letters. 555(2):351-7, 2003).
Neuronal ceroid-lipofuscinosis type 4 (CLN-4) Synonyms: Kufs disease, Kufs-Mayer disease, adult amaurotic idiocy, adult ceroid lipofuscinosis, adult ganglioside lipidosis, adult neuronal ceroid-lipofuscinosis, adult recessive neuronal ceroid lipofuscinosis, chronic adult-recessive neuronal ceroid-lipofuscinosis, late familial amaurotic idiocy late ganglioside lipidosis. A rare variant with onset of symptoms between the ages of 20 and 50 years with a chronic course and associated with cerebellar ataxia, bulbar symptoms, and extrapyramidal and pyramidal signs, but without retinal lesions and rapidly progressive dementia. Transmitted as an autosomal recessive trait but some cases are sporadic. J.-J. Martin, Dev. Neurosci. 13, 331-338, 1991.
Neuronal ceroid-lipofuscinosis type 5 (CLN-5) Synonyms: Boehme disease, Parry neuronal ceroid-lipofuscinosis, adult dominant neuronal ceroid-lipofuscinosis, chronic adult dominant neuronal ceroid-lipofuscinosis, dominant Kufs disease, and dominant neuronal ceroid-lipofuscinosis. It is a cerebellar syndrome with onset early in the fourth decade, characterized by epileptic fits, myoclonic epilepsy, progressive dementia, and hypertension. CLN5 has been shown to be a novel gene encoding a transmembrane protein which is mutated in Finnish variant of LINCL. M. Savukoski, T. Klockars, V. Holmberg, P. Santavuori, E. S. Lander, L. Peltonen, Nat. Genet. 19, 286-288,1998.
Neuronal ceroid-lipofuscinosis type 6 (CLN-6) Synonyms: Zeman-Dyken-Lake-Santavuori-Savukoski disease, and subacute transitional early juvenile neuronal ceroid-lipofuscinosis. A subacute variant with onset in late childhood or in early period with seizures, ataxia, retinal lesions, mental failure, and gradual neurological deterioration. Novel mutations in the CLN6 gene caused a variant, late infantile neuronal ceroid lipofuscinosis (Teixeira C. A., Espinola J., Huo L., Kohischutter J., Persaud Sawin D. A., Minassian B., Bessa C. J., Guimaraes, A., Stephan D. A., Sa Miranda M. C., MacDonald M. E., Ribeiro M. G., Boustany R. M., Human Mutation. 21(5):502-8, 2003). Fine mapping of bovine ceroid lipofuscinosis was confirmed by orthology with CLN6 (Broom M. F., Zhou C., European Journal of Paediatric Neurology. 5 Suppl A:33-5, 2001). Analysis of candidate genes in the CLN6 critical region was also carried out using in silico cloning (Sharp J. D., Wheeler R. B., Schultz R. A., Joslin J. M., Mole S. E., Williams R. E., Gardiner R. M., European Journal of Paediatric Neurology. 5 Suppl A: 29-31, 2001). The loci for classical and late infantile neuronal ceroid lipofuscinosia have been shown to map to chromosomes 11p15 and 15q21-23 (J. D. Sharp, R. B. Wheeler, M. Savukoski, I. E. Jarvela, I. Peltonen, R. M. Gardiner, R. E. Williams, Hum. Mol. Genet., 6,591-595, 1997.
The palmitoyl protein thioesterase-2 (PPT2) gene encodes a lysosomal thioesterase homologous to PPT1. It has been shown that PPT2 deficiency in mice causes an unusual form of neuronal ceroid lipofuscinosis with striking visceral manifestations. In the study cited above (P. Gupta et al., 2001) all PPT2-deficient mice displayed a neurodegenerative phenotype with spasticity and ataxia by 15 months. The bone marrow of such mice was infiltrated by brightly autofluorescent macrophages and multinucleated giant cells. PPT2 deficiency in mice manifests as a neurodegenerative disorder with visceral features. Although PPT2 deficiency has not been described in humans, manifestations would be predicted to include neurodegeneration with bone marrow histiocytosis, visceromegaly, brown pancreas, and linkage to chromosome 6p21.3 in affected families (P. Gupta, A. A. Sombo, J. M. Shelton, I. G. Wilkofsky, K. E. Wisniewski, J. A. Richardson, and S. L. Hofmann, P.N.A.S. USA., 100 (21): 12325-12330, 2003).
The first three Russian cases of classical late-infantile neuronal ceroid lipofuscinosis have been reported (Lavrov, A. Y., Ilyna, E. S., Zakharova, E. Y., Boukina, A. M., Tishkanina, S. V., European Journal of Paediatric Neurology. 6(3): 161-4, 2002). There are also several possible subtypes—protracted, atypical, and earlier or later onset, which have similar clinical symptoms and may become apparent at different ages and progress at different rates. Ultimately, all forms are fatal. In the US, the incidence of all four types may be as high as two or three per 100,000 births. Batten disease is a fatal, inherited disorder of the nervous system that begins in childhood. “Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autonomic excessive neurodegenerative disease caused by mutations in the CLN2 gene (NINDS Batten Disease Information Page at http://www.ninds.nih.govidisorders/batten/batten.htm attached with the parent application as Appendix A).
CLN2 encodes a lysosomal protease that was later found to be identical with lysosomal tripeptidyl peptidase. The specificity of lysosomal tripeptidyl peptidase-1 was determined by its action on angiotensin-II analogues (Warburton M. J., Bernardini F., FEBS Letters, 500(3): 145-8, 2001). It has been shown that the enzyme Palmitoyl protein thioesterase (PPT) localizes into synaptosomes and synaptic vesicles in neurons, and its implications for infantile neuronal ceroid lipofuscinosis (INCL) have been postulated (Lehtovirta, M., Kyttala, A., Eskelinen, E. L., Hess, M., Heinonen, 0., Jalanko, A., Human Molecular Genetics. 10 (1):69-75, 2001). An excellent review on the selectivity and types of cell death in the neuronal ceroid lipofuscinoses has been written (Mitchison H. M., Lim M. J., Cooper J. D., Brain Pathology, 14 (1):86-96, 2004). It was shown that optic nerve degeneration takes place in a murine model of juvenile ceroid lipofuscinosis (Sappington R. M., Pearce D. A., Calkins D. J., Investigative Ophthalmology & Visual Science. 44(9):3725-31, 2003).
At the cellular level, LINCL is characterized by lysosomal accumulation of autoflourescent storage material whose major identifiable component is mitochondria ATP synthase subunit c (subunit c) in neurons and other cell types. Affected individuals usually develop normally until about age 3 years, at which point they exhibit symptoms such as ataxia and seizure. The disease is associated with progressive loss of neurons and photoreceptors, and, within several years (NINDS; Batten Disease Information Page.). LINCL patients become blind, mute, bedridden and demented. Currently, there is no effective treatment for the disease and death typically occurs between age 6 and 15. Early symptoms of this disorder usually appear between the ages of 2 and 4, when parents or physicians may notice a previously normal child who has begun to develop vision problems or seizures (NINDS Batten Disease Information Page). In some cases the early signs are subtle, taking the form of personality and behavior changes, slow learning, clumsiness, or stumbling. Over time, affected children suffer mental impairment, worsening seizures, and progressive loss of sight and motor skills. Eventually, children with Batten disease become blind, bedridden, and demented, and the disease subsequently becomes fatal (NINDS Batten Disease Information Page).
Role of L-Carnitine
From a biochemical standpoint, L-carnitine plays an essential role in energy metabolism. In fatty acid metabolism, it serves as shuttle between the mitochondrial membrane and the mitochondria inner-workings permitting breakdown of the long-chain carbon fragment.
The more important role of L-carnitine is in maintaining a balance between the concentrations of a compound called acyl CoA in the cell compartments. For sugar to be metabolized, they are sequentially degraded to smaller fragments until carbon dioxide is produced, and along the way energy is conserved. Acyl CoA is an important intermediate in transfer of energy. Accordingly, it is important that the concentration of Acyl CoA be regulated and this function falls on L-carnitine. The role of L-carnitine and L-carnitine supplementation during exercise in humans has been illustrated (Brass E. P., Hiatt W. R., J. Am. Coll. Nutr., 17(3):207-215, 1998).
It has also been shown that defects in fatty acid oxidation are a source of major morbidity, particularly among children. Fatty acid oxidation defects encompass a spectrum of clinical disorders, including recurrent hypoglycemic, hypoketotic encephalopathy or Reye-like syndrome in infancy with secondary seizures and potential developmental delay, progressive lipid storage myopathy, recurrent myoglobinuria, neuropathy, and progressive cardiomyopathy. (I. Tein, J Child Neurol. 2002 December; 17 Suppl 3:3 S57-82; discussion 3S82-3).
Supplementation or treatment of a number of these diseases/disorders with L-carnitine has had beneficial effects. For example, some researchers believe L-carnitine supplementation may complement other therapies for the treatment of AIDS. (Effect of L-carnitine on human immunodeficiency virus-1 infection-associated apoptosis; Moretti S., Alesse E., Di Marzio L., a pilot study. Blood, 91(10):3817-3824, 1998). According to the authors, the treatment of immunodeficiency virus type 1 infections, acquired immune deficiency syndrome (AIDS), may elicit or cause carnitine deficiency problems. Additionally, some epileptic patients may benefit from carnitine supplementation or treatment.
L-carnitine may be essential or “conditionally” essential for several groups of people including: normal infants, premature infants, and both children and adults suffering from a variety of genetic, infectious, and injury-related illnesses. For example, some cardiomyopathies, which afflict children, are due to metabolic errors or deficiencies. There is data that supports treatment of some myocardial dysfunctions with L-carnitine supplementation. (Winter, S., Jue, K., Prochazka J., Francis, P., Hamilton, W., Linn, L., Helton, E. (1995) J. Child Neurol. 10, Supple 2: S45-51.)
L-carnitine may also play an essential role in the treatment of several disease conditions. Administration of L-carnitine prevents acute ammonia toxicity and enhances the efficacy of ammonia elimination as urea and glutamine. In addition, the cytotoxic effects of ammonia, possibly arising from lipid peroxidation, are ameliorated by L-carnitine. These data indicate the feasibility of utilization of L-carnitine in the therapy of human hyperammonemic syndromes, both for reducing the levels of ammonia and preventing its toxic effects. (O'Connor J E, Adv Exp Med. Biol. 1990; 272:183-95).
L-carnitine deficiency can be defined as a decrease of intracellular L-carnitine, leading to an accumulation of acyl-CoA esters and an inhibition of acyl-transport via the mitochondrial inner membrane. This may cause disease by the following processes:
A. Inhibition of the mitochondrial oxidation of long-chain fatty acids during fasting causes heart or liver failure. The latter may cause encephalopathy by hypoketonaemia, hypoglycemia and hyper-ammonemia. It was shown that acetyl-L-carnitine fed to old rats partially restores mitochondrial function and ambulatory activity (Hagen T M, Ingersoll R. T, Wehr C. M, Proc. Natl. Acad. Sci. USA., 95(16): 9562-9566, 1998).
B. Increased acyl-CoA esters inhibit many enzymes and carriers. Long-chain acyl-CoA affects mitochondrial oxidative phosphorylation at the adenine nucleotide carrier, and also inhibits other mitochondrial enzymes such as glutamate dehydrogenase, L-carnitine acetyltransferase and NAD transhydrogenase. (Scholte H R, J Clin Chem Clin Biochem. May 1990; 28(5): 35)
C. Accumulation of triacylglycerols in organs increases stress susceptibility by an exaggerated response to hormonal stimuli (lyer, R. N., Khan, A. A., Gupta, A., Vajifdar, B. U., Lokhandwala, Y. Y., J Assoc Physicians India, 8(11):1050-1052, 2000).
D. Effect of L-carnitine on exercise tolerance in chronic stable angina: a multicenter, double-blind, randomized, placebo controlled crossover study (Cherchi, A., Lai, C., Angelino, F., et al., Int. J. Clin. Pharmacol. Ther. Toxicol., 23(10):569-572, 1985).
E. Decreased mitochondrial acetyl-export lowers acetylcholine synthesis in the nervous system.
Primary L-carnitine deficiency can be defined as a genetic defect in the transport or biosynthesis of L-carnitine. Until now, only defects at the level of L-carnitine transport have been discovered. The most severe form of primary L-carnitine deficiency is the consequence of a lesion of the L-carnitine transport protein in the brush border membrane of the renal tubules. This defect causes cardiomyopathy or hepatic encephalopathy usually in combination with skeletal myopathy. In a patient with cardiomyopathy and without myopathy, it was found that L-carnitine transport at the level of the small intestinal epithelial brush border was also inhibited. The patient was cured by L-carnitine supplementation. Muscle L-carnitine increased, but remained too low, suggesting that L-carnitine transport in muscle is also inhibited. L-carnitine transport in fibroblasts was normal, which disagrees with literature reports for similar patients. W. R. Treem, C. A. Stanley, D. N. Finegold, D. E. Hale, P. M. Coates, N. Engl. J. Med, 319, 1331-1336, 1988; G. Karpati, S. Carpenter, A. G. Engel, G. Watters, J., Allen, S. Rothman, G. Klassen, 0. A. Mamer, Neurology, 25, 16-24, 1975; B. O. Eriksson, S. Lindstedt, I. Nordin, Eur. J. Pediatr., 147, 662-663, 1988; H. R. Scholte, R. Rodrigues Periera, P. C. de Jonge, I. E. Luyt-Houwen, M. Verduin, J. D. Ross, J. Clin. Chem. Clin. Biochem. 28, 351-357, 1990; C. A. Stanley, S. DeLeeuw, P. M. Coates, C. Vianey-Liaud, P. Divry, J. P. Bonnefont, J. M. Saudubray, M. Haymond, F. K. Tretz, G. N. Breningstall, Ann. Neuro. 30, 709-716, 1991; Y. Wang, J. Ye, V. Ganapati, N. Longo, Proc. Natl. Acad. Sci. USA 96, 2356-23601999.
In summary, L-carnitine plays a critical role in enhancing fat metabolism. Reports attest to the fact that L-carnitine works by transporting fatty acids to be burned for fuel, increasing both energy supply and lean muscle mass. Most reports also indicate that unless an individual is deficient in L-carnitine, it is an unnecessary ergogenic aid. This contrasts with an apparent need in case of L-carnitine deficiency (e.g., in the case pursued by the inventors of Late Infantile Neuronal Ceroid Lipofuscinosis-one form of Batten Disease), of the correct operation of the endogenous production of L-carnitine. This need was corroborated in the observations of dogs with Batten Disease given exogenous L-Carnitine (Siakotos A. N., Hutchins G. D., Farlow M. R., Katz M. L., European Journal of Paediatric Neurology 5 Suppl A: 151-6, 2001) and those of the parents of the child who was afflicted with LINCL (discussed below).
ATP Dependence on Endogenous L-Carnitine
Optimal ATP production from either dietary or stored fatty acids is dependent on L-carnitine. L-carnitine has several roles, most of which involve conjugation of acyl residues to the b-hydroxyl group of the L-carnitine with subsequent translocation of this complex from one cellular compartment to another. Deficiencies in L-carnitine have been implicated in a number of diseases. For example, in CLN3, proteins have been found to cause modulation of the cell growth rates and apoptosis (Persaud-Sawin D. A., Van Dongen A., Boustany R. M., Human Molecular Genetics. II(18):2129-42, 2002). It has been shown that defects in lysosomal enzymes cause Neuronal Ceroid Lipofuscinoses (NCLs), CLN1 and CLN2. (Hofmann S. L., Atashband A., Cho S. K., Das A. K., Gupta P., Lu J. Y., Current Molecular Medicine. 2(5):423-37, 2002). It has been also shown that the conditions of Parkinson's disease are present when there is dysfunction in both striatal and nigral neurons and this dysfunction results in autosomal dominant adult neuronal ceroid lipofuscinosis (Nijssen, P. C., Brusse, E., Leyten, A. C., Martin, J. J., Teepen, J. L., Roos, R. A., Movement Disorders, 17(3):482-7, 2002). It has been shown that abnormal accumulation of specific proteins occurs in the neuronal ceroid lipofuscinosis/Batten disease. These conditions result due to defect in the lysosomal proteases and related enzymes. The phenomenon is commonly termed as lysosomal proteinoses. This abnormal accumulation of proteins in the lysosomes has been shown to be responsible for major diseases such as Alzheimer disease, alpha-synuclein in Parkinson's disease, Lewy body dementia (Gupta, P., Hofmann, S. L., Molecular Psychiatry. 7(5):434-6, 2002). An autoantibody inhibitory to glutamic acid decarboxylase in the neurodegenerative disorder, Batten disease has been reported (Chattopadhyay, S., Ito, M., Cooper, J. D., Brooks, A. I., Curran, T. M., Powers, J. M., Pearce, D. A., Human Molecular Genetics. 11(12): 1421-31, 2002). Mutations in different proteins result in similar diseases of neuronal ceroid lipofuscinoses (Weimer, J. M., Kriscenski-Perry, E., Elshatory, Y., Pearce, D. A., NeuroMolecular Medicine. 1(2): 111-24, 2002). Lysosomal localization of the neuronal ceroid lipofuscinosis CLN5 protein. (Isosomppi, J., Vesa, J., Jalanko, A., Peltonen, L., Human Molecular Genetics. 11(8):885-91, 2002).
Carnitine Biosynthesis & Metabolism
N-6-Trimethyl-L-Lysine (TML)
The enzyme Tripeptidyl Peptidase 1 (TPP-1) is responsible for cleaving “protein bound N-6-trimethyl-L-lysine” with the resulting products of free TML and amino acids in normal people. However, in children who have Late-Infantile Neuronal Ceroid Lipofuscinoses (LINCL), the TPP-1 is defective and the “protein-bound TML” is not broken down (M. L. Kaz, Biochem. Biophy. Acta, 1317, 192-198, 1996). It therefore becomes the storage material in the lysosome. Eventually it builds up and then consequently “blows up” the lysosome, causing eventual massive neuronal damage in brain and eventually death (P. Gupta and S. L. Hofmann, Molecular Psychiatry, 7, 434-436, 2002).
It has been shown that there is specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with LINCL, and is caused by a defect in the CLN2 gene product, TPP-1. The data by the authors show that TPP-1 is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c (Ezaki, J., Takeda-Ezaki, M., Kominami, E., J Biochem (tokyo) September, 128(3), 509, 2000).
Lysosomal hydrolysis of these proteins results in the release of TML, which is the first metabolite of L-carnitine biosynthesis. Hepatic synthesis of carnitine takes place from protein-bound N-6-trimethyl-L-lysine. Lysosmal digestion of methyl-lysine labeled asialo-fetuin was carried out (LaBadie, J., Dunn, W. A. and Aronson Jr, N. N. Biochem. J. 160, 85-95,1976). L-carnitine biosynthesis has been studied, such as, from gamma-butyrobetaine and from exogenous protein-bound-6-N-trimethyl-L-lysine by perfused guinea pig liver. In this connection, the effect of ascorbate deficiency on the in situ activity of gamma-butyrobetaine hydroxylase was demonstrated (Dunn, W. A., Rettura, G., Seifter, E. and Englard, S., J. Biol. Chem. 259, 10764-10770, 1984).
TML to L-Carnitine Pathway
TML is first hydroxylated on its 3-position to form 3-hydroxy-N-6-trimethyl-L-lysine (HTML). The aldolytic cleavage of HTML with HTML Aldolase (HTMLA) yields trimethylaminobutyraldehyde (TMABA) and glycine. Dehydrogenation of TMABA by TMABA dehydrogenase (TMABA-DH) results in the formation of 4-N-trimethylaminobutyrate (butyrobetaine). In the last step, gamma-butyrobetaine is hydroxylated on the 3 position by gamma-butyrobetain deoxygenase (BBD; EC to yield L-carnitine (Frederic M. Vaz and Ronald J. A. Wanders, Biochem. J. 361, 417-429, 2000). Very little is known about HTMLA. It might be identical to serine and lycine hydroxymethyltransferase (SHMT) which catalyses the tetrahydrofolate-dependent interconversion of serine and glycine (Girgis, S., Nasrallah, I. M., Suh, J. R., Oppenheim, E., Zanetti, K. A., Mastri, M. G. and Stover, P. J., Gene 210, 315-324, 1998). Purification and characterization of cytosolic and mitochondrial serine hydroxymethyltrasferase from rat liver was carried out (Ogawa, H. and Fujioka, M. J. Biochem. (Tokyo) 90, 381-390, 1981). SHMT also catalyses the aldol cleavage of other beta-hydroxylamino acids in absence of tetrahydrofolate, including HTML (Girgis, S., Nasrallah, I. M., Suh, J. R., Oppenheim, E., Zanetti, K. A., Mastri, M. G. and Stover, P. J. Gene 210, 315-324, 1998). Synthesis of butyrobetaine and L-carnitine from protein bound TML is inhibited by 1-amino-D-proline, an antagonist of vitamin B6. This inhibitory effect of 1-amino-D-proline on the production of L-carnitine from exogenous protein-bound N-6-trimethyl-L-lysine by the perfused rat liver has been shown. (Dunn, W. A., Aronson Jr, N. N. and Englard, S., J. Biol. Chem. 257, 7948-7951, 1982).
It is well known from the biochemistry of the metabolic pathway of TML to HTML that certain cofactors; such as 2-oxoglutarate, Fe2+, molecular oxygen and ascorbate, have to be present. Similarly in the subsequent steps of metabolic pathway from HTML to L-carnitine, the biochemically defined cofactors have to be present. The cofactors (2-oxoglutarate, Fe2+, molecular oxygen, and ascorbate) have been established by a number of researchers during the enzymatic hydroxylation of TML. It is likely that other chemicals will work as cofactors as well. For example, DTT (dithiothreitol) has been used instead of ascorbic acid (which is required to keep Fe2+ in reduced form), in test tube conditions. Besides the aforesaid cofactors, calcium ion was found to cause significant enhancement in the conversion of TML to HTML (D. S. Sachan, C. L. Hoppel, Biochem. J., 188, 529-534, 1980).
4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) catalyzes the dehydrogenation of 4-N-trimethylamino butyraldehyde to butyrobetaine. TMABA-DH has an absolute requirement for NAD+. In human tissues, the rate of TMABA dehydrogenation is highest in liver, substantial in kidney, but low in brain, heart and muscle (Rebouche, C. J. and Engel, A. G., Biochim. Biophys. Acta, 22-29, 1980). TMABA-DH has been purified from beef liver (Hulse, J. D. and Henderson, L. M., Fed. Proc. Fed. Am. Soc. Exp. Biol., 38, 676, 1979).
Gamma-butyrobetaine dioxygenase (BBD) catalyses the stereospecific hydroxylation of butyrobetaine to L-carnitine in mammalian studies. BBD activity was stimulated considerably by 2-oxoglutarate, and the enzyme requires molecular oxygen, Fe2+ and ascorbate for activity. (Lindblad, B., Lindstedt, G. and Tofft, M., J. Am. Chem. Soc., 91, 4604-4606, 1969). BBD activity has been found to be localized in the cytosol.
Kakimoto and Akazawa were the first to identify TML in human urine. All methods to assay TML in either plasma, urine or tissue samples use the same sample work-up. The concentration of TML in plasma is relatively constant in both human and rat, ranging from 0.2 to 1.3 micromole. Plasma levels of TML have been shown to correlate with body mass. In humans, urinary TML concentration is proportional to that of creatine. Furthermore, TML is not reabsorbed by kidney in humans. (Davis, A. T., Ingalls, S. T. and Hoppel, C. L J. Chromatogr. 306, 79-87, 1984.). In humans, TML concentrations range between 2 to 8 micromole per mmole of creatine. (Kakimoto, Y. and Akazawa, S., J. Biol. Chem. 245, 5751-5758, 1970).
Butyrobetaine is the last step in the synthesis of L-carnitine. The level of butyrobetaine in urine is low (about 0.3 micromole/mmol creatinine) (F. M. Vaz, B. Melegh, J. Bene, D. Cuebas, D. A. Gage, A Bootsma, P. Vreken, A. H. van Gennip, L. L. Bieber and R. J. A. Wanders, unpublished work) compared with the concentration in plasma of 4.8 micromole (Sandor, A., Minkler, P. E., Ingalls, S. T. and Hoppel, C. L., Clin. Chim. Acta., 176, 17-27, 1988).
Factors in the Biosynthesis & Control of L-Carnitine and N-6-Trimethyl-L-Lysine
Major sources of L-carnitine in the human diet are meat, fish and dairy products. Omnivorous humans generally ingest 2-12 micromoles of L-carnitine per day per kg of body weight. This is more than the L-carnitine produced endogenously, which has been estimated to be 1.2 micromole per day per kg of body weight. In omnivorous humans, approximately 75% of body L-carnitine sources come from the diet and 25% come from de novo biosynthesis. Since L-carnitine is present primarily in foods of animal origin, strict vegetarians obtain <0.1 micromole per day per kg of body weight. Strict vegetarians obtain more than 90% of their L-carnitine through biosynthesis.
Two primary intermediates have been proposed as the factors which limit biosynthesis of L-carnitine via their availability. These two intermediaries are g-butyrobetaine and N-6-trimethyl-L-lysine. Studies have shown that increasing the amount of either of these two intermediates in the bloodstream will increase the production of L-carnitine 100-fold in rats and 3-fold in human infants and adults (Olson and Rebouche, J. Nutr. 117(6), 1024-31,1987). Thus, L-carnitine biosynthesis may be regulated by one or all of the three enzymes which, together, catalyze the transformation of N-6-trimethyl-L-lysine into g-butyrobetaine. The high level of L-carnitine synthesis from exogenous L-carnitine precursors suggests that the enzymatic capacity to synthesize L-carnitine from TML and butyrobetain is much higher than is usually utilized. This suggests that only the availability of TML is the rate limiting step in the regulation of feedback inhibition for L-carnitine biosynthesis (Schematic 1). (F. M. Vaz and R. J. A. Wandars, Biochem. J., 361, 417-429, 2002).

L-ascorbic acid may be a principle co-factor in the metabolism of L-carnitine. It has been postulated and demonstrated that an experimental vitamin C deficiency resulted in increased urinary excretion of L-carnitine. This increased excretion of L-carnitine may be due to either decreased absorption from dietary sources, or increased excretion from the kidney. Several methods have been described to measure the concentration of L-carnitine biosynthesis metabolites in biological fluids and tissues.
The kidney plays a major role in L-carnitine biosynthesis, excretion and acylation. Unlike in the rat, the human kidney contains the enzymes needed to form L-carnitine from N-6 trimethyl-L-lysine (K, Doqi, National Kidney Foundation. Am. J. Kidney Dis., 35, 6 Suppl 2 S1-140, 2000). This L-carnitine precursor, TML, is found to be increased in plasma of patients with chronic renal failure. Free L-carnitine formed in the kidney as well as L-carnitine reabsorbed from the glomerular filtrate may be acylated in the proximal tubule. Isolated rat cortical tubule suspensions contain total L-carnitine concentrations of 2.85 micromols/g protein. During incubation over 60 min, the acylcarnitine/carnitine ratio decreased, indicating deacylation of acylcarnitine in proximal tubules. Exogenous L-carnitine was acylated at a rate of 35 micromols/h/g protein. Besides pyruvate and acetate, ketone bodies stimulated the acylation rate several fold, indicating that these substrates are a major source of acetyl-CoA for the acylation reaction. This may explain the higher acetylcarnitine/L-carnitine ratio found in urine under ketotic conditions.
However, later data shows that the brain also participates in active synthesis of L-carnitine from TML (F. M. Vaz and R. J. A. Wandars, Biochem. J., 361, 417-429, 2002). The concentration of butyrobetaine in plasma and tissues was determined by isolating butyrobetaine via HPLC or ion-exchange chromatography, and using BBD to convert it into L-carnitine. In humans, the level of butyrobetaine in urine is low (about 0.3 micromole/mmole L-creatinine) compared with the concentrations in plasma (4.8 mmole & 1.8 mmole).
The concentration of L-carnitine in plasma from both humans and rats is age and sex dependent. In humans, the plasma L-carnitine concentration increases during first year of life (from about 0.15 to about 0.40 mmole) and remains the same for both sexes until puberty. From puberty to adulthood, plasma L-carnitine concentrations in males increases and stabilizes at a level that is significantly higher than those in females (50 micromole compared to 40 micromole).
Obviously, carnitine is available from exogenous sources (meat, milk). However, work has been done to see if exogenous carnitine would ameliorate the symptoms of Juvenile Neuronal Ceroid Lipofuscinosis, not LINCL. This research in dogs showed that it made the dogs more functional and they lived 10% longer than untreated dogs, but the dogs still died very young compared to unaffected dogs and brain glucose hypometabolism and cerebral atrophy were not reduced (Siakotis, Katz et al., European Journal Pediatric Neurology 5, (Suppl. A): 151-156, 2001). It is an exciting prospect to see that TML may indeed be that therapeutic agent to cause positive brain metabolism based upon the results we have seen.
This invention provides a method of exogenic supplementation with TML to affect L-carnitine biosynthesis, thereby influencing ATP levels.